The term “menotropins” is applied to a hormonal combination obtained from menopausal and post-menopausal women's urine comprising two glycoprotein hormones: follicle stimulating hormone and luteinizing hormone. These two hormones are secreted by the pituitary gland, and subsequently metabolized and excreted in the urine.
Menotropins and follitropin have been long used in the therapeutical treatment of infertility disorders.
The role of FSH consists in acting on the ovarian follicles, promoting their rapid growth and maturity with subsequent ovulation or atresia. FSH along with LH is also involved in the biosynthesis of estradiol in the ovarian follicles that it has stimulated. The internal theca is the main site of androgen follicular biosynthesis which is under the control of LH. Controlled by FSH, this androgen is then aromatized in the granulose cells and excreted to the blood torrent. Therefore, LH is a necessary constituent along with FSH in follicular stimulation.
Pituitary hormones FSH and LH. thyroid stimulating hormone (TSH) and the placental hormone human chorionic gonadotropin (HCG) are closely related since all of them are glycoproteins having in their structure two subunits called α and β. Both subunits are bound by non-covalent interaction. These subunits do not have any biological activity separately. The α-chains of the four above-mentioned hormones are common, while the β-chains are different and provide a biological activity characteristic of each hormone. However, important portions of the β-chain are also common, which can be particularly observed in LH and HCG β-chains.
Gonadotropins obtained from human urine have been partially purified until reaching biosafety characteristics compatible with the requirements of an injectable pharmaceutical product, and have been commercialized for more than 30 years in order to solve infertility problems; being in the form of an injectable grade pharmaceutical preparation, they are listed in the most important pharmacopoeias in the world and have been approved for pharmaceutical use in practically all countries all over the world.
The composition of the follitropin preparation can be described as follows:    Follicle stimulating hormone: 75 or 150 International units of FSH    Excipient (required for the lyophilization process, generally 5–20 mg of lactose) and other non-active proteins.
The composition of the menotropin preparation can be described as follows:    Follicle stimulating hormone: 75 or 150 International units of FSH    Luteinizing hormone 75 or 150 International units of LH    Excipient (required for the lyophilization process, generally 5–20 mg of lactose) and other non-active proteins.
International units of FSH and LH are calculated by measuring the biological activities in rats relative to an international standard prepared by the National Institute for Biological Standards and Control (NIBSC) dependent on the World Health Organization.
The requirement, imposed by Pharmacopoeias such as United States Pharmacopoeia (USP XXIII) or British Pharmacopoeia (BP) in terms of purity of the starting material required for the preparation of injectable grade menotropins, consists in a starting material having an FSH activity greater than 40 IU/mg and an LH activity greater than 40 IU/mg. In view of this, those starting materials heretofore used have been obtained by a manufacturing process ensuring an activity in the range of 70–150 IU FSH/LH per mg., which is more than enough to meet the requirements imposed by the health organizations as far as biosafety and effectiveness are concerned.
However, the latest developments in terms of purification techniques and the development of gonadotropins from recombinant origin have influenced the need for preparations of highly purified native gonadotropins of urinary origin, without the presence of impurifying proteins.
Some FSH and LH purification process have been described in the recent past.
EP 0 322 438 B1 and the equivalent U.S. Pat. Nos. 5,128,453; 5,767,067 and 5,840,857 refer to the preparation of follicle stimulating hormone with high specific activity from urinary origin. However these processes include a specific monoclonal antibodies step. From the safety point of view, this fact raises some concerns about contamination of the product with heterologous proteins, DNA residues from the hosts cell and viruses. Although process validations and final testing can help to exclude the possibility of potential contamination, it is reasonable to judge that a process that is specially designed to avoid using biological reagents has higher safety standards than the one which includes this type of purification step.
The WO 98/20039 patent application describes a FSH and LH separation and purification process which avoids employing biological reagents. However, due to the very high FSH:LH ratio of the raw material used and products obtained, this process excludes the possibility of obtaining menotropins, a preparation which requires an FSH:LH ratio of approximately 1:1
Moreover, WO 98/20039 fails to characterize the gonadotropins obtained as a bioactive material, which is an essential issue for a therapeutical product. This patent application uses an immunoradiometric method to test the activity, a determination method that arises deep concerns about its accuracy.
In fact, it has been long recognized that the structural heterogeneity of FSH and LH isoforms has an important impact on the biological activity and immunological reactivity of both hormones [Costagliola et. al. J. Endocrinol. Invest. 17, 291 (1994)]. Observed differences in bio- and immuno-FSH and LH levels suggest that separated structural entities are recognized by the bioassays versus the immunoassay. It has been thoroughly discussed that immunoassays does not consistently provide a good estimation of the bioactive gonadotropins level and does not necessarily reflect the biological activity [Dahl and Stone, J. Andrology, 13, 11 (1992); Rose et. al Endocrine reviews 21, 5 (2000)].
On the other hand, bioassays are considered to be the proper test to define the hormone activity since it takes into account two important components that are absent in others methods: the biological action at the target tissue and the biological clearance [Rose et. al. Endocrine reviews 21, 5 (2000)]. This fact makes the in vivo bioassay mandatory for calibration of therapeutics preparations [Rose, Clinica Chimica Acta 273, 103 (1998)].
It must be also underlined that the WO 98/20039 fails also to characterize the gonadotropins as a chemically pure drug since no analytical method is presented to support this fact.
The present invention presents a full characterization of the gonadotropins not only from the bioactivity scope but also from the analytical point of view by introducing sensitive analytical method to test the purity. Since the assays used to establish the FSH and LH activity in the present invention are, in all the cases, in vivo bioassays which has been long proved to be a robust specific test for assuring bioactivity, the potency of the FSH and the LH for each gonadotropins obtained and also their ratio can be assured.
A second issue to consider is that the above mentioned patent (WO 98/20039) describes the use of an affinity dye chromatography to purify gonadotropins. This procedure could arise some concern about the potential contamination of the product by the leakage of the dye. These kind of foreign compounds are not desirable in products to be injected into humans [Protein Purification by R. K. Scopes, Springler-Verlag New York Inc; 2nd Edition, page 156 (1998)]. Since the present invention was design to exclude this type of chromatography, the dye contamination problem is an issue completely avoided.
Current menotropins (the commercially available product) may suffer the drawback of potential local allergic reactions when they are administered subcutaneously, due to the presence of protein contaminants.
This invention provides a method for the purification and the preparation of the first commercially available high purity gonadotropin products. This methodology was specially designed so as to avoid using biological reagents such as antibodies, receptros and other heterologus materials and chromatography dyes. Besides the safety issue, the use of non-specific steps during the isolation allows to obtain of all the isoforms that are present in the starting material.
As extensively described, heterogeneity is of particular importance in the glycoprotein hormones. At least 20–30 different isoforms of FSH, LH and TSH exist (Wide, Acta Endocr., Copenh 109, 181–189, 1985). These isoforms differ in their molecular weight and charge. Although glycoprotein hormone isoforms mainly vary in the oligosaccharide structure, microheterogeneities may be also present from differences in the amino-acid composition (Costagliola et al, J. Endocrinol. Invest., 17, 291–299, 1994; Wilson et al., J. Endocrinol 125, 3–14, 1990).
It is generally accepted that the type of different isoforms isolated depends, among other factors, on the isolation techniques (Cockburn et al., Biologicals 19, 257–264, 1991). The use of highly discriminating techniques for isolation, such as monoclonal antibodies, can contribute to select only one part of the isoforms present in the source of the material. Being “too specific”, some biologically active gonadotropin variants may not be recognized by the antibodies and could be lost (Costagliola et al, J. Endocrinol. Invest., 17, 291–299, 1994). On the other hand, a non-selective approach, such as the one described in this patent, can be useful in purifying all the types of isoforms present in the urine. Different isoforms have shown to vary with respect to the interaction with the cell surface receptors and metabolic clearance (Thotakura et al., Glycobiology 5, 3–10, 1995). Oligosaccharide structure is under the control of various physiological factors. From the therapeutical point of view, it is interesting to have a product in which all isoforms present in the urine are available. In this way, the highly purified products described here provide the differential roles of naturally occurring gonadotropin isoforms in the maintenance of reproductive function.
It must also be underlined that the present application is the first characterization of a highly purified menotropins preparation that was confirmed not only by its high biological potency but also by relevant screening methods (PAGE electrophoresis, HPLC, size exclusion chromatography).
Being now available in a purified grade and in a large scale, these naturally occurring FSH and LH molecules can be then investigated and carefully analyzed. The three dimensional structure of the hormones can contribute to a better understanding about the role of the oligosaccharides.
The method for obtaining currently produced menotropins is well known. As indicated in BP 1980, menotropins can be prepared from post-menopausal women's urine by kaolin adsorption, subsequent alkali elution and precipitation with 2 volumes of acetone. The required fraction is extracted from this precipitate with an ammonium acetate solution in 70% ethanol and then precipitated with a 10% ammonium acetate solution in 90% ethanol. The menotropins product is obtained by ionic exchange chromatography of this precipitate.
The starting material, from now on the “HMG source material”, that can be used in the present invention for obtaining highly purified menotropins, constitutes the menotropins specialty, as specified in the pharmacopoeias (USP XXIII, BP 1993 addendum 1995; EP 1986) or any other material closely related to this specialty. Therefore it is understood that this preparation process is also applicable to other materials that do not strictly meet the requirements applicable to menotropins. In fact, satisfactory results were obtained using Fraction C (see description of the invention. Example 1 and example 2), which is a material that falls to comply with the FSH:LH ratio menotropins requirement (FSH:LH, 1:1, approximately) Moreover, the present invention provides a procedure to adjust the FSH:LH ratio when needed being therefore capable of providing highly purified menotropins specialty even when starting materials which are out of the correct FSH:LH ratio are used. Thanks to this, it is possible to obtain a product comprised of FSH and LH in the appropriate ratio 1:1 necessary to produce the pharmaceutical preparation, without having separated both active principles during purification. That is to say, accomplishing the co-purification of both hormones, from a purity degree of approximately 5% in the starting material to more than 95% in the final product with a final potency 25–35 times greater than the initial one. The purification degree as obtained can be visualized from FIG. 1, which shows the result of polyacrylamide gel electrophoresis of the conventional pharmaceutical product vs. the new purified version obtained from the application of the present Invention.
The steps described in the present invention can be used following in a different order from the one herein described, which does not imply varying the invention philosophy. Equally satisfactory results were obtained by inserting, for example, the hydrophobic interaction resin between the two ionic exchange chromatography steps described later.
This invention also provides for the preparation of urinary gonadotropins separately, that is to say FSH and LH.
The administration of menotropins compositions for therapeutic purposes has been carried out, mainly by intramuscular injection. This administration form creates a considerable discomfort in the patient and requires from the patient regular visits to clinical units, sometimes for weeks or months in order to receive the treatment.
Subcutaneous administration would make the self-administration possible and consequently improve patient's cooperation and compliance.
The subcutaneous administration of urinary gonadotropins has already been described (Nakamura Y., et al, Fertil Steril 51, 423–429, 1989; Engmann L. et al. Fertil Steril. 69, 836–840, 1998). The subcutaneous administration of non-pure preparations may suffer the drawback of local allergies due to the presence of impurities in the product used and consequently result in the suspension of the treatment. Therefore it is worthwhile to produce high purity products which can diminish the possibility of these allergic reactions.